DESCRIPTION (provide by applicant): The overall goal of this research is to understand the role of protein fatty acylation and membrane cholesterol in directing subcellular localization and function of signal transduction proteins. Members of the Src family of tyrosine protein kinases will be used as model systems. Specifically, the experiments outlined in this application are designed to address the following: 1. To identify signaling molecules and pathways activated by plasma membrane cholesterol depletion. We recently showed that depletion of membrane cholesterol with cyclodextrin activates MAP kinase and causes increased tyrosine phosphorylation on a number of proteins. The identity of these proteins will be determined, as well as the relationship(s) between MAPK activation and protein tyrosine phosphorylation. The intracellular signaling pathways that are activated will be identified using pharmacological and dominant negative protein inhibitors. 2. To determine the kinetics and regulation of Src family kinase (SFK) depalmitoylation. Palmitoylation is a dynamic reaction that controls protein localization and signaling. We will determine whether APT1, a newly described thioesterase, depalmitoylates SFKs and whether SFK depalmitoylation is regulated by raft localization and/or signal transduction. 3. To identify the intracellular trafficking mechanism(s) utilized by Src family kinases. The subcellular trafficking pathway(s) involved in directing SFKs to specific membrane locations will be determined using combinations of confocal microscopy and time-lapse imaging of GFP-tagged SFKs. We will test whether SFKs interact with components of the endocytic machinery en route to the plasma membrane.